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Table 1 Dynamic cultivations performed with the recombinant P. pastoris strain carrying a novel bi-directional promoter system

From: A novel bi-directional promoter system allows tunable recombinant protein production in Pichia pastoris

Experiment

Substrate

Feeding strategy based on qs [g g−1 h−1]a

Goals

FB1

Glycerol

Batch on glycerol

Fed-batch on glycerol with step-wise decrease of qs,gly

0.567–0.450–0.282–0.142–0.101–0.070–0.050 g g−1 h−1 (each step was done for 2 h)

Methanol pulses: 0.5% (v/v) adaptation pulse at 30 °C—two 2% (v/v) pulses at 30 and 20 °C, respectively

Determination of strain specific physiological parameters (qs,max,gly; qs,gly for maximum qp; qs,adapt,MeOH; qs,max,MeOH)

Characterization of recombinant expression profile at different process parameters

FB2

Glycerol and methanol

Batch on glycerol

0.5% (v/v) methanol adaptation pulse

Constant MeOH feed at qs 0.02–0.025 g g−1 h−1 and concomitant glycerol feed with stepwise increases: 0.025-0.059-0.139-0.239 g g−1 h−1 (each step was done for 2 h)

Find operating window for maximum qp

FB3

Glycerol and methanol

Batch on glycerol

Fed-batch at qs,gly = 0.212 g g−1 h−1

0.5% (v/v) methanol adaptation pulse followed by a fed-batch on methanol at qs = 0.034 g g−1 h−1

Mixed feed cultivation at qs,gly = 0.135 g g−1 h−1 and qs,MeOH = 0.035 g g−1 h−1

Demonstrate tunability of recombinant protein production

  1. aThe qs values were adjusted based on our findings during this study (vide infra)